Melatonin action on luteal - granulosa cells in women with marital infertility undergoing in vitro fertilization

Introduction of the subject: Melatonin is a hormone related to the light-dark cycle and it plays an influential role in the reproductive system. In humans, researchers have shown there is three times more melatonin in follicular fluid than in the blood flow, suggesting the influence of the hormone on follicular maturation. However, little is known about melatonin action on granulosa cells in women with infertility, especially the molecular mechanisms involved. We are, therefore, conducting this study to evaluate melatonin action pathways in granulosa cells in the ovaries of women with infertility. To achieve our aim, we will use molecular biology, involving diverse signaling pathways, such as angiogenesis. Objective(s): to analyze melatonin action on luteal - granulosa cells in women with marital infertility undergoing in vitro fertilization. Methodology: For this, 68 patients, aged between 20 and 35 years, attended at the Human Reproduction Sector of the Federal University of São Paulo, were submitted to in vitro fertilization treatment. After all preparatory procedures were performed, luteal granulosa cells were removed and routed to the cell culture. The cells were divided into four groups: a) control; b) 0.1 μM melatonin; c) 1 μM melatonin; d) melatonin at 10μM. After a period of 10 days, the cells were trypsinized for extraction of the total RNA and later analysis of the gene expression by Real Time PCR of the angiogenesis signaling pathway. Results: Our data evaluated 96 genes, which are related to the angiogenesis pathway. The results of transcriptional expression showed important genes involved in this pathway that are hypo or hyperexpressed after treatment with melatonin. The main hyperexpressed genes were: fibroblast growth factor 1 (FGF1) genes; interleukin 1-beta (IL1B); receptor tyrosine kinase (VEGFR-2); folliculogenesis regulating genes (TGFB1). They act in the dynamics of follicular growth. On the other hand, the genes inducing factor 1-alpha (HIF1A), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGFA) melatonin decreased its expression. Discussion and conclusion of the results: It is concluded that melatonin in high concentration (above 1 μM) may have dual function in increasing the expression of some growth factors and cytokines, in the decrease of other genes and mechanism of physiological compensation and also modulates negative angiogenesis, mainly in the dose (10 uM) of granulosa cells from women submitted to in vitro fertilization. Perhaps this fact is important for adequate follicular growth, avoiding excessive growth, which could turn the follicle into cyst, making ovulation difficult.Keywords: Melatonin; Granulosa cells; Women; Marital infertility; In vitro fertilization

Effects of Pentoxifylline on TNF-Alpha and Lung Histopathology in HCl-Induced Lung Injury

OBJECTIVE: To evaluate the effects of pentoxifylline on hydrochloric acid-induced lung lesions in rats subjected to mechanical ventilation. METHODS: Twenty male, adult Wistar-EPM-1 rats were anesthetized and randomly grouped (n=5 animals per group) as follows: control-MV (mechanical ventilation, MV group); bilateral instillation of HCl (HCl group); bilateral instillation of HCl followed by pentoxifylline (50 mg/kg bw) infusion (HCl+PTX group) and pentoxifylline infusion followed by bilateral instillation of HCl (PTX+HCl group). At 20, 30, 90 and 180 min after treatments, the blood partial pressures of CO2 and O2 were measured. The animals were euthanized, and bronchoalveolar lavages were taken to determine the contents of total proteins, corticosterone and TNF-alpha. Samples of lung tissue were used for histomorphometric studies and determining the wet-to-dry (W/D) lung weight ratio. RESULTS: In the MV group, rats had alveolar septal congestion, and, in the HCl group, a remarkable recruitment of neutrophils and macrophages into the alveoli was noticed; these events were reduced in the animals with PTX+HCl. The partial pressure of oxygen increased in PTX+HCl animals (121±5 mmHg) as compared with the HCl (62±6 mmHg) and HCl+PTX (67±3 mmHg) groups within 30 minutes. TNF-alpha levels in bronchoalveolar lavage were significantly higher in the HCl group (458±50 pg/mL), reduced in the HCl+PTX group (329±45 pg/mL) and lowest in the PTX+HCl group (229±41 pg/mL). The levels of corticosterone in bronchoalveolar lavage were significantly lower in the HCl (8±1.3 ng/mL) and HCl+PTX group (16±2 ng/mL) and were highest in the PTX+HCl (27±1.9 ng/mL). CONCLUSION: Pretreatment with PTX improves oxygenation, reduces TNF-alpha concentration and increases the concentration of corticosterone in bronchoalveolar lavage upon lung lesion induced by HCl

Morphology of the interstitial cells of rat polycystic ovaries: an experimental study

PURPOSES: To evaluate the histomorphometry of ovarian interstitial cells, as well as the blood sex steroid concentrations of female rats with polycystic ovaries induced by continuous light. METHODS: Twenty female rats were divided into two groups: Control Group - in the estrous phase (CtrlG), and a group of rats with polycystic ovaries induced by continuous illumination (POG). CtrlG animals were maintained on a light period from 07:00 a.m. to 07:00 p.m., and POG animals with continuous illumination (400 Lux) for 60 days. After this period all animals were anesthetized and blood was collected for the determination of serum estradiol (E2), progesterone (P4), and testosterone (T), followed by removal of the ovaries that were fixed in 10% formalin and processed for paraffin embedding. Five-µm histological sections were stained with hematoxylin and eosin and used for histomorphometric analysis. Morphological analyses, cyst count, determination of concentration and of the nuclear volume of interstitial cells were performed with the aid of a light microscope adapted to a high resolution camera (AxioCam), whose images were transmitted to and analyzed by the computer using AxioVision Rel 4.8 software (Carl Zeiss). Data were analyzed statistically by the Student's t-test (pCtrlG=73.2±6.5, pCtrlG=80.6±3.9, pPOG=4.2±1.5, pCtrlG=63.6±16.5, pCtrlG=6.9±3.2, pGCtrl=73,2±6,5; pGCtrl=80,6±3,9; pGOP=4,2±1,5; pGCtrl=63,6±16,5; pGCtrl=6,9±3,2; p<0,05) em relação aos animais do GCtrl. CONCLUSÃO: As células intersticiais do ovário policístico da rata provavelmente provêm dos cistos ovarianos devido degeneração das células da granulosa e diferenciação das células da teca interna. As elevações dos níveis séricos de testosterona e de estradiol provavelmente provêm do aumento significativo da atividade celular e da área ocupada pelas células intersticiais.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Morfologia e GenéticaUniversidade de São Paulo Faculdade de Medicina Departamento de Obstetrícia e GinecologiaUNIFESP, EPM, Depto. de Morfologia e GenéticaSciEL

Evaluation of FAS and caspase-3 in the endometrial tissue of patients with idiopathic infertility and recurrent pregnancy loss

Objective: To investigate expression of BcL-2, FAS, FAS ligand (FASL) and cleaved caspase-3 in the endometrial tissue of women with idiopathic infertility (with two consecutive failed cycles of in vitro fertilization) and women with idiopathic recurrent pregnancy loss. the control group consisted of fertile women.Study design: Endometrial tissue samples from fertile women (n = 25), women with idiopathic infertility (n = 25) and women with idiopathic recurrent pregnancy loss (n = 25) were collected on the seventh or eighth postovulatory day of their menstrual cycles for evaluation. Expression of BcL-2, FAS, FASL and cleaved caspase-3 was assessed using immunohistochemical methods.Results: Expression of BcL-2 and FAS was significantly higher and lower, respectively, in the women with idiopathic infertility than in the other groups (p < 0.01). Expression of cleaved caspase-3 was significantly lower in the women with idiopathic infertility than in the other groups (p < 0.01). Expression of FASL was similar in all three groups.Conclusion: Disturbances in endometrial apoptosis may be a contributing factor in patients with idiopathic infertility and recurrent pregnancy loss. (C) 2012 Elsevier Ireland Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Gynaecol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Obstet, São Paulo, BrazilUniv São Paulo, Dept Obstet & Gynaecol, Gynaecol Div, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Gynaecol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Obstet, São Paulo, BrazilFAPESP: 2004/01424-0Web of Scienc

Assessing the benefits of rosiglitazone in women with polycystic ovary syndrome through its effects on insulin-like growth factor 1, insulin-like growth factor-binding protein-3 and insulin resistance: a pilot study

Federal University of Sao PauloFederal University of ParaibaCAPE

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation

OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip¯ Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip¯ Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis

Effects of melatonin on the ovarian gene expression in pinealectomized rats

Objetivo: Este estudo pretende identificar os genes alvo da melatonina no tecido ovariano de ratas adultas pinealectomizadas. Material e Métodos: Foram estudadas 32 ratas (Rattus norvegicus albinus), adultas virgens, procedentes do Biotério da UNIFESP – EPM. Após confirmação da ciclicidade estral, os animais foram divididos em dois grupos: GI- pinealectomizado recebeu veículo; GII - pinealectomizado com reposição de melatonina (10 μg/noite/rato), durante 60 dias consecutivos. Ao final da ministração, todos os animais foram anestesiados e os ovários coletados foram colocados imediatamente em nitrogênio líquido por meia hora e em seguida congelado a - 80°C para análise das amostras por cDNA “microarray”. Para a determinação da expressão dos diferentes genes, foi utilizado o Kit GeneChip® Rat Genome 230 2.0 Array da Affymetrix, de acordo com as especificações do fornecedor, repetindo-se o experimento três vezes para cada grupo. Os dados obtidos foram normalizados e submetidos ao programa GeneChip® Operating Software. Posteriormente, confirmados pelo software de análise secundária do DNA-Chip Analyzer (dChip). Foram considerados como significantes quando estava 1,5x aumentados (hiperexpressos) ou diminuídos (hipoexpressos) em relação ao veículo. Além disso, foram escolhidos genes relacionados com a função ovariana que foram confirmados pela técnica de RT-PCR. Resultados: Nossos dados mostraram que GII (tratado com melatonina) teve 101 genes hiperexpressos e 72 hipoexpressos em comparação ao GI (recebeu melatonina). Em relação à esteroidogênese e de significância estatística, os genes hiperexpressos: inibina beta-A (INHBA), folistatina (FST), Abl-Interactor 1 e, hipoexpressos: Sintetase da prostaglandina D2 (Brain), LIM Homeobox 9, Glutathiona S-Transferase Mu 3. No RT-PCR, confirmamos os resultados da inibina e da folistatina que estão relacionados com a função ovariana. Conclusão: Nossos dados sugerem que melatonina poderia interferir na expressão gênica nos ovários de ratas pinealectomizadas, principalmente na hiperexpressão da inibina e da folistatina.BV UNIFESP: Teses e dissertaçõe